1. Field of the Invention
The present invention relates generally to the field of molecular biology. More specifically, the invention relates to methods and compositions for more efficiently transforming cells.
2. Description of the Related Art
Agrobacterium sp., including A. tumefaciens, are soil borne phytopathogenic bacteria that cause crown gall disease in plants. This disease is a manifestation of the transfer, integration and expression of oncogenes on a specific region of the transferred DNA (T-DNA) in susceptible hosts (Amand and Mysore, 2005; Gelvin, 2003; Tzfira and Citovsky, 2006). The mechanism utilized by A. tumefaciens and related species to transfer T-DNA into plant cells also allows engineering of plants with engineered DNA, for instance comprising a transgene of interest.
In addition to the T-DNA, several Agrobacterium encoded proteins, such as VirD2, VirE2, VirE3 and VirF are also translocated into plants (Christie, 2004; Cascales and Christie, 2004; Vergunst et al., 2003). It is thought that A. tumefaciens separately translocates the VirD2-T-strand and VirE2 and that the VirD2-T-strand-VirE2 complex (T-complex) assembles in the plant cell (Cascales and Christie, 2004; Vergunst et al., 2000). VirD2 remains tightly attached to the 5′ end of the nicked T-DNA region, while the remaining single stranded DNA (ssDNA) is covered stoichiometrically with VirE2, protecting the T-strand from exonucleolytic degradation in planta.
The T-complex is subsequently imported into the nucleus most likely through interactions with other host proteins such as VIP1 (Tzfira et al., 2001) and importin α (Ballas and Citovsky, 1997). Once inside the plant cell, the T-complex is stripped of its proteins possibly through targeted proteolysis involving the SCFvirF ubiquitin complex (Tzfira et al., 2004). The T-DNA most-likely relies on host DNA repair machinery for its conversion into double stranded (ds) T-DNA intermediates and their recognition by proteins like KU80 (Li et al., 2005) and histone H2A (Li et al., 2005; Mysore et al., 2000) for integration into the host chromosome.
Several plant proteins that interact with the T-DNA nucleoprotein complex have been identified, for instance via yeast two-hybrid assays. These include VIP1 (U.S. Patent Publ. 20030233676, GenBank AF225983; Tzfira et al., 2001) and VIP2 (Tzfira and Citovsky, 2000; GenBank AF295433; GenBank AK117230; GenBank DQ000202; GenBank BG130671). However, the mechanism by which VIP2 acts in relation to Agrobacterium-mediated DNA transfer has not been known. Further, methods to utilize VIP2 to enhance such DNA transfer have also not been known.